Ancrod-specific monoclonal antibodies, antibody fragments, mixtures or derivatives thereof and use of the same

ABSTRACT

Ancrod-specific monoclonal antibodies, antibody fragments, mixtures or derivatives thereof are used in pharmaceutical preparations and in diagnosis. Cells which express these antibodies, antibody fragments, mixtures or derivatives thereof are also disclosed.

FIELD OF THE INVENTION

The invention relates to ancrod-specific monoclonal antibodies, antibodyfragments, mixtures or derivatives thereof and their use inpharmaceutical preparations or in diagnosis, and to pharmaceuticalpreparations which comprise these antibodies, antibody fragments,mixtures or derivatives thereof.

The invention furthermore relates to cells which express theseantibodies, antibody fragments, mixtures or derivatives thereof.

BACKGROUND OF THE INVENTION

Ancrod (proprietary name: Arwin®, Arvin®) is an enzyme from the venom ofthe Malayan pit viper (Agkistrodon rhodostoma). It is a highlyglycosylated serine protease which has an average MW of about 38000 andwhich has anticoagulant properties and the ability to dissolve bloodclots.

Normal coagulation of blood is effected by thrombin which eliminatesfibrinopeptides A and B from the fibrinogen molecule and thus leads tothe formation of fibrin (EP-B-0 556 906), the main constituent ofthrombi in addition to, for example, red blood corpuscles or platelets.In contrast to thrombin, acrod cleaves only the arginine-glycine linkagein the a(“A”) chain of the fibrinogen molecule, which liberatesfibrinopeptides A, AP and AY (Cole et al., J. Vascular. Surgery, Vol 17,1993: 288-292). The β(B) chain of the fibrinogen molecule is notattacked by ancrod and is thus not liberated. The fragments(de-“A”-fibrin monomers) produced after the elimination of thefibrinopeptides caused by ancrod are eventually able to polymerize tothin filaments. The resulting atypical, soluble fibrin is lyzed byendogenous plasmin and/or removed by the reticuloendothelial system(=RES, monocyte/macrophage system). Further cleavage of thede-“A”-fibrinogen molecule by thrombin to give natural fibrin no longertakes place because the resulting molecule is not a thrombin substrate.

Ancrod causes a dose-dependent decrease in the blood fibrinogenconcentration. Therapeutically induced and controlled hypofibrinogenemiadiminishes the plasma viscosity and tendency of erythrocytes toaggregate so far that the flow properties of the blood are cruciallyimproved. This provides the condition for greater flow of blood throughstenosed vessels. Ancrod is currently used to treat, for example,chronic disturbances of peripheral arterial blood flow, and isundergoing clinical phase III studies on stroke.

Ancrod is advantageously injected subcutaneously. Treatment can takeplace in hospital or, if the regular checks of the fibrinogenconcentration necessary to monitor the therapy are ensured, also on anoutpatient basis. Intravenous administration of ancrod is possible butshould take place only in exceptional cases and under hospitalobservation.

The dosage of ancrod must also be individualized. The behavior of thefibrinogen concentration as a function of the ancrod dose is crucial. Itmust be slowly reduced to 70-100 mg/100 ml of plasma (=therapeuticrange). The fibrinogen concentration must be adjusted to be within thisrange throughout the treatment period. The flow properties of the bloodare satisfactory under these conditions. The therapy normally lasts 3-4weeks but can, if necessary, be extended beyond this period.

On subcutaneous administration, 70 I.U. (=international units, 1 ml) aregiven each day in the first 4 days, and 70-140 I.U. are given, dependingon the behavior of the fibrinogen concentration, from day 5 onwards. Ifthe fibrinogen concentration is in the therapeutic range, singleinjections of 210-280 I.U. are given 2-3 times a week.

On intravenous infusion, initially 2-3 I.U./kg of body weight are givenover the course of eight hours. The subsequent dosage of ancrod dependson the fibrinogen concentration attained. It is generally sufficient toinject a further 1 I.U./kg of body weight slowly every 12 hours.

The initial half-life of ancrod in the circulation is about 3-5 hours,but slows down as the concentration falls so that after about 4 days,within this time in general 90% of the administered ancrod areeliminated, the half-life is extended to 9-12 days.

Although ancrod contrasts with, for example, heparin and warfarin inbeing associated with fewer problems of unspecific bleeding during thetreatment (see Z. S. Latallo, “Retrospective Study on Complications andAdverse Effects of Treatment with Thrombin-Like Enzymes—A MulticenterTrial”, Thromb. Haemostasis, 50 (1983) 604-609), specific treatment ofsuch bleeding is necessary and desirable.

Contraindications for treatment with ancrod are, for example,hemorrhagic diathesis, danger of bleeding associated with injuries,after operations and deliveries, for ulcerative intestinal disorders,neoplasms, poorly controllable hypertension, acute cerebral infarct andactive pulmonary tuberculosis, dysfunctions of the RES and disturbancesof clot breakdown, eg. in states of high fever, severe liver disorders,manifest and incipient states of shock or pregnancy.

As described above, the risk of bleeding is relatively low with ancrodwhen the fibrinogen concentration is reduced slowly and is adjusted to70-100 mg/100 ml during the period of therapy. Patients with a latenttendency to bleed, eg. cases of kidney stones or renal failure, shouldbe monitored particularly carefully. Arterial punctures andintramuscular injections of other drugs should be avoided. Caution isnecessary on concurrent administration of RES-blocking and ulcerogenicdrugs, anticoagulants, antifibrinolytics, thrombolytics and medicineswhich inhibit platelet aggregation, and on intramuscular administrationof ancrod. Absorption from the muscle depot generally takes place veryquickly so that too many de-“A”-fibrin monomers flow away and there is adanger of thromboembolic complications.

The total incidence of bleeding in a study on 429 patients (Crit. Rev.Oncol. Hematol. 15 (1993) 23-33), who received ancrod without previousthrombolytic therapy was 9.8% (4.2% internal bleeding; 5.6% externalbleeding).

Currently used to neutralize the enzymatic activity of ancrod is anantidote based on an immunoglobulin preparation from goat serum (KnollAG publication, June 1983, entitled Arwin®). This antidote consisting ofpolyclonal antibodies is used in cases of severe hemorrhagiccomplications or increased danger of bleeding, eg. associated withaccident injuries or because surgery suddenly becomes indicated.Neutralization of ancrod should be followed by administration of 4-5 gof human fibrinogen. If human fibrinogen, plasma or blood isadministered without previous neutralization of ancrod by an antidote,there is a danger of acute disseminated coagulation.

Stocker et al (Thrombosis Research, Vol. 6, 1975: 189-194) investigatedthrombogenesis in the presence of Arwin® alone and in the presence ofthe polyclonal antidote and were able to demonstrate the antidoteeffect.

Besides this use of polyclonal antibodies from goats, EP-B-0 395 375,EP-B-0 556 906 and Burkhardt et al (FEBS, Vol 297, No. 3, 1992: 297-301)describe monoclonal or polyclonal antibodies for detecting expression ofancrod genes, for detecting fibrinogen in blood using ancrod and ancrodantibodies or purification of ancrod using antibodies.

A disadvantage of the goat polyclonal antibodies used as ancrod antidoteis that, for example, they consist of a mixture of antibodies, many ofwhich have no ancrod-neutralizing effect. This large number of differentantibodies may lead to a rapid immune response and, moreover, leads to arelatively low ancrod-neutralizing capacity. In addition, the antidotecontains antibodies of varying affinity for ancrod. Polyclonalantibodies can, because they are obtained from animals, be standardizedonly with difficulty, which means that there are variations in thedifferent production batches.

SUMMARY OF THE INVENTION

It is an object of the present invention to develop an antidote toancrod which does not have the abovementioned disadvantages and is easyto produce industrially.

We have found that this object is achieved by the novel monocloncalantibodies, antibody fragments, mixtures or derivatives thereof whichbind to ancrod and inhibit its activity, where the binding affinity isin a range from 1×10⁻⁷ to 1×10⁻¹² M, and the neutralizing effect isimproved at least 100% by comparison with goat polyclonal antibodies invivo.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph depicting the quantification of ancord (proprietaryname Arwin) by optical density with antibodies Mab 1-2 and Mab 3-27.

DETAILED DESCRIPTION OF THE INVENTION

The novel antibodies used as ancrod antidote advantageously have anumber of improved properties. For example, they form a homogeneous,well-characterized product consisting of one antibody or one antibodysubclass showing no variations between different production batches.They can be produced in any desired quantity, and production thereofdoes not entail any risk of viral or bacterial contamination becausethey are not produced in animals. The novel antibodies, antibodyfragments, mixtures or derivatives thereof are epitope-specific and showa high binding and neutralizing activity. They can therefore beadministered in small amounts for treatment. The homogeneity of theproduct together with the smaller amounts used, owing to the highbinding and neutralizing activity, result in a marked reduction in therisk of an immune response in the patient. Variations in the binding andneutralizing activity like those with polyclonal antibodies do not occurwithin the various antibodies, antibody fragments or derivatives. Mixingdifferent monoclonal antibodies, antibody fragments or derivatives withbinding activity for different epitopes of ancrod allows the latter tobe neutralized very efficiently.

The novel antibodies, antibody fragments, mixtures or derivativesthereof advantageously have a binding affinity for ancrod in a rangefrom 1×10⁻⁷ to 1×10⁻¹² M, preferably from 1×10⁻⁸ to 1×10⁻¹¹,particularly preferably from 1×10⁻⁹ to 5×10⁻¹⁰ M.

The novel antidote has an ancrod-neutralizing effect in vivo which isimproved, by comparison with goat polyclonal antibodies, by at least100%, preferably by 250%, particularly preferably by 500%. Themonoclonal antibodies also show a distinctly better effect than thepolyclonal antibodies in vitro.

Novel monoclonal antibodies or fragments thereof mean in principle allimmunoglobulin classes such as IgM, IgG, IgD, IgE, IgA or theirsubclasses such as the IgG subclasses or mixtures thereof. IgG and itssubclasses are preferred, such as IgG₁, IgG₂, IgG_(2a), IgG_(2b), IgG₃or IgG_(M). The IgG subtypes IgG₁/κ and IgG_(2b)/κ are particularlypreferred. Fragments which may be mentioned are all truncated ormodified antibody fragments with one or two antigen-complementarybinding sites which show high binding and neutralizing activity towardancrod, such as parts of antibodies having a binding site whichcorresponds to the antibody and is formed by light and heavy chains,such as Fv, Fab or F(ab′)₂ fragments, or single-stranded fragments.Truncated double-stranded fragments such as Fv, Fab or F(ab′)₂ arepreferred. These fragments can be obtained, for example, by enzymaticmeans by eliminating the Fc part of the antibody with enzymes such aspapain or pepsin, by chemical oxidation or by genetic manipulation ofthe antibody genes. It is also possible and advantageous to usegenetically manipulated, non-truncated fragments.

The antibodies or fragments can be used alone or in mixtures.

The antibody genes for the genetic manipulations can be isolated, forexample from hybridoma cells, in a manner known to the skilled worker.For this purpose, antibody-producing cells are cultured and, when theoptical density of the cells is sufficient, the mRNA is isolated fromthe cells in a known manner by lyzing the cells with guanidiniumthiocyanate, acidifying with sodium acetate, extracting with phenol,chloroform/isoamyl alcohol, precipitating with isopropanol and washingwith ethanol. cDNA is then synthesized from the mRNA using reversetranscriptase. The synthesized cDNA can be inserted, directly or aftergenetic manipulation, for example by site-directed mutagenesis,introduction of insertions, inversions, deletions or base exchanges,into suitable animal, fungal, bacterial or viral vectors and beexpressed in appropriate host organisms. Preference is given tobacterial or yeast vectors such as pBR322, pUC18/19, pACYC184, lambda oryeast mu vectors for the cloning of the genes and expression in bacteriasuch as E. coli or in yeasts such as Saccharomyces cerevisiae.

The invention furthermore relates to cells which synthesize the novelantibodies. This [sic] may be animal, fungal, bacterial cells or yeastcells after transformation as mentioned above. They are advantagoeuslyhybridoma cells or trioma cells, preferably hybridoma cells. Thesehybridoma cells can be produced, for example, in a known manner fromanimals immunized with ancrod and isolation of their antibody-producingB cells, selecting these cells for ancrod-binding antibodies andsubsequently fusing these cells to, for example, human or animal, forexample, mouse mylemoa cells, human lymphoblastoid cells orheterohybridoma cells (Koehler et al., Nature 256, 1975: 496) or byinfecting these cells with appropriate viruses to give immortal cells.Hybridoma cell lines produced by fusion are preferred, mouse hybridomacell lines are particularly preferred, and hybridoma cell lines whichsecrete the antibodies MAb 1-2, MAb 2-29/3 or MAb 3-27 and which havebeen deposited at the DSMZ (Deutsche Sammlung für Mikroorganismen undZellkulturen in Braunschweig) under the numbers DSM ACC2317, DSM ACC2318and DSM ACC2319 are very particularly preferred.

The abovementioned hybridoma cell lines secrete particularly preferredantibodies of the IgG type. The antibodies MAb 1-2, MAb 2-29/3 and MAb3-27 which are formed are of the IgG subtypes IgG₁/κ, IgG_(2b)/κ andIgG₁/κ. These preferred antibodies bind to different epitopes of theancrod molecule, as has been shown by tests on competitive binding ofthe antibodies among themselves. The binding of the particularlypreferred antibody MAb 1-2 to its epitope results in the most extensiveneutralization of the ancrod molecule, and thus the smallest amounts ofantibody are required to neutralize the enzymatic effect. The monoclonalantibodies show a distinctly greater neutralizing effect than theantidote which is normally used for treating hemorrhages and is based onpolyclonal antibodies which are obtained from goats and are marketed byKnoll AG (Ludwigshafen) as antidote.

Derivatives of the novel monoclonal antibodies which may be mentionedhere are peptides, peptidomimetics derived from the antigen-bindingregions of the antibodies, and antibodies, fragments or peptides boundto solid or liquid carriers such as polyethylene glycol, glass,synthetic polymers such as polyacrylamide, polystyrene, polypropylene,polyethylene or natural polymers such as cellulose, Sepharose oragarose, or congugates with enzymes, toxins or radioactive ornonradioactive markers such as 3H, 123I, 125I, 131I, 32P, 35S, 14C,51Cr, 36Cl, 57Co, 55Fe, 59Fe, 90Y, 99mTc, 75Se, or antibodies, fragmentsor peptides covalently bonded fluorescent/chemiluminescent labels suchas rhodamine, fluorescein, isothiocyanate [sic], phycoerythrin,phycocyanin, fluorescamine, metal chelates, avidin, streptavidin orbiotin.

The novel antibodies, antibody fragments, mixtures and derivativesthereof can be used directly, after drying, for example freeze drying,after attachment to the abovementioned carriers or after formulationwith other pharmaceutical active and ancillary substances for producingpharmaceutical preparations. Examples of active and ancillary substanceswhich may be mentioned are other antibodies, antimicrobial activesubstances with a microbiocidal or microbiostatic action such asantibiotics in general or sulfonamides, antitumor agents, water,buffers, salines, alcohols, fats, waxes, inert vehicles or othersubstances customary for parenteral products, such as amino acids,thickeners or sugars. These pharmaceutical preparations are used tocontrol diseases, preferably to control coagulation disturbances,advantageously disturbances of the peripheral blood system, or forstroke.

The novel antidote can be administered orally orparenterally—subcutaneously, intramuscularly, intravenously orinterperitoneally [sic]—and intramuscular or intravenous administrationis preferred.

The novel antibodies, antibody fragments, mixtures or derivativesthereof can be used in diagnosis directly or after coupling to solid orliquid carriers, enzymes, toxins, radioactive or nonradioactiven labelsor to fluorescent/chemiluminescent labels as described above. In whichcase [sic] ancrod can be detected in a wide variety of body fluids froma wide variety of organisms such as humans or animals or a wide varietyof liquids such as culture media from yeasts, bacteria, fungi or humanor animal cell cultures.

EXAMPLES

1. Preparation of the Hybridoma Cell Lines

The immunization, fusion, selection and characterization were carriedout by techniques described in the literature (eg. J. H. Peters;Monoklonale Antikörper, Herstellung und Charakterisierung; SpringerVerlag; A. M. Campbell; Monoclonal Antibody and Immunosensor Technology;published by Elsevier, chapters 2 to 7 and 8, 1991).

Female Balb/c mice were immunized intraperitoneally with 100 μg ofancrod which had been inactivated in respect of enzymatic activity bycrosslinking, in a 2-3 week rhythm in accordance with the followingadministration scheme:

1. in 100 μl of PBS+100 μl of complete Freund's adjuvant

2. in 100 μl of PBS+100 μl of incomplete Freund's adjuvant

3.-5. in 200 μl of PBS

Three days after the last antigen administration, the spleen wasremoved, the cells were washed and isolated, and the lymphocytes werefused to the myeloma cell line SP2/0-Ag14 (=ATCC CRL 1581). This wasdone by mixing them in the ratio of 5:1, incubating with 1.5 ml of PEGsolution (=polyethylene glycol solution at 37° C. for 1 min and mixingwith PBS (=phosphate-buffered saline) (1 ml for 30 sec., 3 ml for 30sec., 16 ml for 60 sec.). After a washing step, the cells werecultivated in selection medium [DMEM (=Dulbecco's Modified EagleMedium); 10% FCS (=fetal calf serum); 10% Condimed H1 (BoehringerMannheim); HAT supplement (=hypoxanthine, aminopterin, thymidinesupplement); ITS supplement (=insulin, transferrin, selenitesupplement); pyruvat; glutamine; streptomycin/penicillin] at 37° C./7.5%CO₂.

Hybridomas specifically secreting anti-ancrod antibodies were identifiedas follows by means of a specific ELISA in a microtiter plate:

coat microtiter plates with 0.1 ml/well ancrod or reference proteins todetermine the specifity (1 μg/ml 0.05 M NaHCO₃ pH 9.2) at 4° C. for 16 h

saturate with 0.3 ml/well 1% BSA/PBS at 23° C. for 0.5-1 h (h=hour)

wash 3× with PBS/0.05% Tween® 20

incubate with cell culture supernatant (50 μl diluted with 50 μl ofPBS/0.1% BSA [=bovine serum albumin)/0.05% Tween® 20) at 23° C. for 2-4h

wash as above

incubate with 0.1 ml/well biotinylated anti-mouse IgG antibody in 0.1%BSA/PBS at 23° C. for 2-4 h

wash as above

incubate with 0.1 ml/well streptavidin-peroxidase complex in 0.1%BSA/PBS at 23° C. for 0.5 h

wash as above

0.1 ml/well peroxidase substrate

stop the reaction with 0.1 ml/well 2 M H₂SO₄

measure the absorption at 450 nm

Peroxidase substrate: mix 0.1 ml of TMB solution (42 mMtetramethylbenzidine in DMSO) and 10 ml of substrate buffer (0.1 M Naacetate pH 4.9) then add 14.7 μl of H₂O₂.

Hybridomas with a positive antibody reaction were isolated by subcloningand the individual clones were retested. Antibodies with the highestreactivity were employed in the in vitro neutralization assay. It waspossible in this way to isolate a large number of positive hybridomas,ie. cells which produce antibodies against ancrod.

2. Production and Characterization of the Monoclonal Antibodies

The monoclonal antibodies were purified from serum-free cell culturesupernatants. This was done by transferring the hybridomas stepwise fromDMEM/HAT/10% FCS medium via DMEM/HT/10% FCS and DMEM/10% FCS into aserum-free cell culture medium (HT=hypoxanthine, aminopterin) such asSF-3 (Cytogen), PFHM-II (Gibco), HL-1 (Bio Whittaker), Ultra Doma PF(Bio Whittaker) or the like. Protein A-Sepharose and Protein G-Sepharosewere used for the subsequent purification by affinity chromatography.

After the cell culture supernatants had been loaded on thechromatography columns, the nonspecifically bound proteins were washedout with 3 M NaCl/1.5 M glycine pH 8.9; the anti-ancrod antibodyreactivity was eluted with 500 mM NaCl/0.59% acetic acid.

The antibody subtype was determined in an ELISA similar to thatdescribed above but using, in place of the biotinylated anti-mouse IgGantibody, the following biotinylated subtype-specific antibodies:anti-mouse IgG₁, anti-mouse IgM, anti-mouse IgG_(2a), anti-mouse κ,anti-mouse IgG_(2b), anti-mouse λ and anti-mouse IgG₃.

The antibody types and subtypes of the isolated hybridoma cell lines MAb1-2, MAb 2-29/3 and MAb 3-27 (see Example 1) were respectivelydetermined as follows IgG₁/κ, IgG_(2b)/κ and IgG₁/κ.

The affinity constants of the monoclonal antibodies were determined byvarious techniques disclosed in the literature, such as, for example,eg. [sic] equilibrium dialysis, immunoprecipitation or ELISA (eg. J. H.Peters; Monoklonale Antikörper, Herstellung und Charakterisierung;Springer Verlag; A. M. Campbell; Monoclonal Antibody and ImmunosensorTechnology; Verlag Elsevier, chapter 11, 1991).

The ELISA method (J. Immunol. Methods 77 (1985) 305-319) revealed thefollowing affinities for natural ancrod (Table I):

TABLE I Affinities of the monoclonal antibodies for ancrodHybridoma/antibody k_(D) MAb 1-2 1.7 * 10⁻⁹ MAb 2-29/3 3.1 * 10⁻⁹ MAb3-27 4.4 * 10⁻¹⁰

3. “In vitro” Neutralization of Ancrod By Monoclonal Antibodies (=MAbs)Cell Culture Supernatants

The neutralizing capacity of the antibodies was quantified by means ofthe acrod-induced fibrin turbidity. This was done by incubating ancrodand antibodies (cell culture supernatants or purified antibodies) invarious ratios of concentrations in BSA-saturated microtiter plates at37° C. and then adding human fibrinogen (1.5 mg). After incubation at37° C., the fibrin which was produced was quantified at 340 nm (=opticaldensity=OD).

The ancrod activity could be neutralized as the amount of neutralizingantibodies increased. This was shown by the decreased optical densities(=OD, Table II).

The antibody MAb 1-2 was particularly effective and resulted in completeneutralization even with relatively high ancrod concentrations (TableII, OD corresponds to the blank). The blank in Table II contained allthe constituents apart from ancrod. The highest OD values were measuredin each case with the various ancrod doses (50, 25 and 12.5 ng/mlancrod) without addition of the various monoclonal antibodies (TableII).

MAbs 2-29 and 3-27 have affinities for ancrod which are as good as orbetter than that of MAb 1-2 (Table I), but the antibody -antigen bindingresulted in neutralization of the enzymatic activity only with higherantibody doses relative to the amount of ancrod.

TABLE II Neutralization of ancrod by cell culture supernatants of theMAbs: Mixtures Optical density (= OD) Blank 0.268 Ancrod 50 ng/ml 1.45Ancrod 50 ng/ml + MAb 1-2 0.254 Ancrod 50 ng/ml + MAb 2-29 1.354 Ancrod50 ng/ml + MAb 3-27 1.133 Ancrod 25 ng/ml 0.939 Ancrod 25 ng/ml + MAb1-2 0.238 Ancrod 25 ng/ml + MAb 2-29 0.333 Ancrod 25 ng/ml + MAb 3-270.422 Ancrod 12.5 ng/ml 0.67 Ancrod 12.5 ng/ml + MAb 1-2 0.229 Ancrod12.5 ng/ml + MAb 2-29 0.281 Ancrod 12.5 ng/ml + MAb 3-27 0.261

4. “In vitro” Neutralization of Ancrod By Purified Monoclonal Antibodies

It was possible to evaluate the in vitro neutralizing efficiency of thepurified monoclonal antibodies by comparing the 50% neutralizationvalues in the fibrin turbidity assay.

The 50% neutralization value was obtained as follows:

 OD negative control+(OD positive control−OD negative control)/2

Negative control:no ancrod added (no fibrin formation)

Positive control:ancrod+fibrinogen (maximum fibrin formation)

Different antibody/ancrod ratios resulted in varying OD values, and the50% neutralization point was reached with the following antibodyconcentrations:

Polyclonal Polyclonal Ab/MAb MAb 1-2 antibody ratio Preincubation for 1hour 1.25 ng of ancrod 310 ng 625 ng 2.0 2.5 ng of ancrod 350 ng 800 ng2.3 Preincubation for 2 hours 1.25 ng of ancrod  80 ng 310 ng 3.9 2.5 ngof ancrod 150 ng 650 ng 4.3

It was posible to deduce from the ratios of the amount of antibodyrequired for 50% neutralization that, under the chosen in vitroconditions, the monoclonal antibody MAb 1-2 is better by at least afactor of 2 with preincubation for 1 hour, and by a factor of about 4with preincubation for 2 hours, than the antidote based on goatpolyclonal antibodies.

5. Quantification of Ancrod By Means of a Sandwich ELISA

Ancrod was be [sic] determined in samples for diagnostic purposes, eg.various body fluids, by means of a sandwich ELISA with a combination oftwo antibodies as shown in the following scheme:

coat the microtiter plates with 5 μg/ml MAb 1-2 or MAb 3-27 in 100μl/well, diluted in 0.05 M NaHCO₃, pH 9.2; 4° C. overnight

wash the microtiter plates with PBS/0.05% Tween® 20;

200 μl/well

saturate with 300 ml/well 1% BSA/PBS; 23° C. for 0.5 h

wash as above

11 standard 2-fold dilutions of ancrod starting with 50 ng/ml inPBS/0.1% BSA/0.05% Tween® 20; 100 μl/well; the samples are employed inparallel in various dilutions; incubate at 23° C. for 2 h

wash as above

incubate with MAb 2-29; 1 μg/ml diluted in PBS/0.1% BSA/0.05% Tween® 20;100 μl/well; 23° C. for 2

wash as above

incubate with biotinylated anti-mouse IgG_(2b); diluted 1:10000 inPBS/0.1% BSA/0.05% Tween® 20; 100 μl/well; 23° C. for 2 h

wash as above

incubate with streptavidin-peroxidase complex, diluted 1:10000 inPBS/0.1% BSA/0.05% Tween® 20; 100 μl/well; 23° C. for 0.5 h

wash as above

add 100 μl/well peroxidase substrate: (mix 10 ml of substrate buffer(0.1M sodium acetate pH 4.9) with 100 μl of TMB solution (42 mMtetramethylbenzidine in DMSO) and add 14.7 μl of 3% H₂O₂)

stop the reaction with 100 μl/well 2 M H₂SO₄

measure the absorption at 450 nm

It emerged that ancrod is quantifiable and detectable in a concentrationrange from about 3000 to 100 pg/ml with both the monoclonal antibodiesMAb 1-2 and MAb 3-27 and the combinations used. The absolute detectionlimit is below these quantifiable values (FIG. 1).

6. Competitive ELISA

A competitive ELISA was carried out to characterize the relativeposition of the MAb binding epitopes on ancrod:

coat microtiter plates with 1 μg/ml ancrod 100 μl/well, diluted in 0.05M NaHCO₃, pH 9.2; 4° C. overnight

wash the microtiter plates with PBS/0.05% Tween® 20; 200 μl/well

saturate with 300 μl/well 1% BSA/PBS; 23° C. for 0.5 h

wash as above

10 ng/ml of each of the biotinylated monoclonal antibodies MAb1-2-biotin, MAb 2-29/3-biotin and MAb 3-27-biotin were placed in variousmixtures in the microtiter plates prepared in this way, and bound toancrod, and then in each case a different antibody (MAb 1-2, MAb 2-29/3or MAb 3-27) was added in various concentrations (1 μg/ml-1 ng/ml)depending on the initial antibody to the mixture so that all possibleantibody combinations were tested for possible overlaps of their bindingsites. The mixtures with the various antibody combinations wereincubated in PBS/0.1% BSA/0.05% Tween® 20 at 23° C. for two hours andthe treated as follows:

wash as above

incubate with streptavidin-peroxidase complex, diluted 1:10000 inPBS/0.1% BSA/0.05% Tween® 20; 100 μl/well; 23° C. for 0.5 h

wash as above

add 100 μl/well peroxidase substrate: (mix 10 ml of substrate buffer(0.1M sodium acetate pH 4.9) with 100 ml of TMB solution (42 mMtetramethylbenzidine in DMSO) and add 14.7 μl of 3% H₂O₂)

stop the reaction with 100 μl/well 2 M H₂SO₄

measure the absorption at 450 nm

No decrease in the OD was observed in any of the antibody combinationsemployed, which means that the various monoclonal antibodies did notdisplace one another on binding to ancrod. They bind to differentepitopes on the ancrod molecule. It is therefore possible for more thanone of the antibodies to interact with ancrod simultaneously. Thevarious novel monoclonal antibodies can therefore, if necessary andrequired, be used in combination for rapid and optimal neutralization ofthe effect of ancrod.

4. In vivo neutralization of ancrod

Ancrod was administered to anesthetized rats by an infusion of 10 IU/kgof body weight into the tail vein for 30 minutes. 10 minutes afterstarting the ancrod infusion, the various test substances—monoclonal,polyclonal antibodies or placebo—were administered as an intravenousbolus of 1 ml/kg of body weight. Blood samples (8 vol. of blood+2 vol.of 0.11 M citrate anticoagulant) were taken from the carotid arterybefore and 30 and 60 minutes after starting the ancrod infusion. Theplasma was obtained from the citrated blood by centrifugation, and thefibrinogen content was determined by the Clauss coagulation method(calibration plot obtained by adding defined amounts of rat fibrinogento fibrinogen-free rat plasma).

6 rats were used in each (test substance) group (Table III).

TABLE III Test substances and amounts used: MAb 1-2 (1.435 mg/kg of bodyweight) polyclonal Ab (8.6 mg/kg of body weight; antidote batch A009)polyclonal Ab (1.5 mg/kg of body weight; antidote batch A009)

TABLE IV Measurement of the fibrinogen concentration with the variousantibodies Fibrinogen conc. [mg/dl] Ancrod + Ancrod + Ancrod + Ancrod +polycl. Ab polycl. AK Time [min] control MAb 1-2 8.6 mg/kg 1.5 mg/kg 0289.7 263.4 292.4 281.8 30 63.8 155.9 129.0 91.5 60 32.1 152.7 164.055.8

It emerged that MAb 1-2 was able, in the concentration of 1.435 mg/kg ofbody weight used, to stop a further decline in the fibrinogen level 30minutes after starting the ancrod infusion. 8.6 mg/kg of body weight arerequired for the same effect with goat polyclonal antibodies. In aconcentration of 1.5 mg/kg of body weight, ie. comparable to theconcentration of MAb 1-2, the antidote based on polyclonal antibodiesshowed no effect (see control in Table IV).

It was possible to deduce on the basis of the fibrinogen concentrationsresulting after 60 minutes that the in vivo neutralization under thetest conditions by MAb 1-2 was a factor of about 6 better than that bythe polyclonal antidote. The neutralizing effect of MAb 1-2 is presumedto be even greater.

We claim:
 1. A monoclonal antibody, antibody fragment, mixture orderivative thereof, which binds to ancrod and inhibits its activity,said monoclonal antibody having a binding affinity of at least 1.7×10⁻⁹M and a 50% neutralization value of about 310 ng as determined in afibrin turbidity assay with a pre-incubation of 1.25 ng ancrod for 1hour.
 2. A monoclonal antibody, antibody fragment, mixture or derivativethereof as claimed in claim 1, wherein the antibody is of the IgG class.3. A monoclonal antibody, antibody fragment, mixture or derivativethereof as claimed in claim 1, which is the antibody MAb 1-2, MAb 2-29/3or MAb 3-27 or mixture thereof.
 4. A cell which expresses a monoclonalantibody, antibody fragment, mixture or derivative thereof as claimed inclaim
 1. 5. A cell as claimed in claim 4, which is from a hybridoma cellline.
 6. A cell as claimed in claim 4, wherein a hybridoma cell line isDSM ACC2317, DSM ACC2318, or DSM ACC2319.
 7. A pharmaceuticalpreparation comprising a monoclonal antibody, antibody fragment, mixtureor derivative thereof as claimed in claim
 1. 8. Compositions fortreating coagulation disturbance in a patient undergoing Ancrod therapycomprising a monoclonal antibody, antibody fragment, mixture orderivative thereof as claimed in claim
 1. 9. A process for diagnosing adisease caused by inappropriate ancrod concentration in body fluids,said process comprising the step of detecting ancrod levels in bodyfluids directly or after coupling to solid or liquid carriers, enzymes,toxins, radioactive or nonradioactive labels or to fluorescent orchemiluminescent labels a monoclonal antibody, antibody fragment,mixture or derivative thereof as claimed in claim
 1. 10. A process asclaimed in claim 9 wherein said step of detecting the ancrod is made inculture media.